1. Field of the Invention
The present invention relates to a method for producing L-glutamic acid. L-glutamic acid is widely used as a raw material for seasonings and so forth.
2. Brief Description of the Related Art
L-Glutamic acid is primarily produced by fermentation utilizing L-glutamic acid-producing bacteria, including coryneform bacteria belonging to the genus Brevibacterium, Corynebacterium or Microbacterium, or mutant strains thereof (Kunihiko Akashi et al., Amino Acid Fermentation, Japan Scientific Societies Press [Gakkai Shuppan Center], pp. 195-215, 1986). Methods for producing L-glutamic acid by fermentation using other microorganisms have also been reported and methods for producing L-glutamic acid using a microorganism belonging to the genus Bacillus, Streptomyces, Penicillium or the like have been reported in U.S. Pat. No. 3,220,929. Methods for producing L-glutamic acid using a microorganism belonging to the genus Pseudomonas, Arthrobacter, Serratia, Candida, or the like have been reported in U.S. Pat. No. 3,563,857. Methods for producing L-glutamic acid using a microorganism belonging to the genus Bacillus, Pseudomonas, Serratia, Aerobacter aerogenes (currently referred to as Enterobacter aerogenes), or the like have been reported in JP32-9393B. Methods for producing L-glutamic acid using a mutant strain of Escherichia coli have been reported in JP5-244970A. In addition, methods for producing L-glutamic acid using a microorganism belonging to the genus Klebsiella, Erwinia, Pantoea, or Enterobacter have been reported in U.S. Pat. No. 6,197,559, U.S. Pat. No. 6,331,419 and European Patent Publication No. 0999282).
Furthermore, methods for enhancing the activities of L-glutamic acid biosynthetic enzymes using recombinant DNA techniques to increase L-glutamic acid-producing ability have been disclosed. For example, it has been reported that the L-glutamic acid-producing ability of Corynebacterium or Brevibacterium bacteria (JP7-121228B) could be effectively improved by introducing a gene encoding citrate synthase derived from Escherichia coli or Corynebacterium glutamicum. Furthermore, it has also been reported that the L-glutamic acid-producing ability of enterobacteria belonging to the genus Enterobacter, Klebsiella, Serratia, Erwinia, or Escherichia (European Patent Publication No. 0999282) could be effectively improved by introducing a citrate synthase gene derived from a coryneform bacterium.
Methods of improving the abilities of bacteria to produce substances such as amino acids by modifying an uptake or export system of the substances are known. As a method of modifying an uptake system for a substance, for example, a method of eliminating or reducing cellular uptake of a substance is known. Specifically, improving an L-glutamic acid-producing ability by eliminating or reducing cellular uptake of L-glutamic acid by deleting the gluABCD operon or a part thereof (European Patent Publication No. 1038970), or by reducing cellular uptake of the purine nucleotide for enhancing a purine nucleotide-producing ability (European Patent Publication No. 1004663), and so forth are known.
Methods for modifying an export system include enhancing an export system of an target substance and eliminating or reducing an export system of an intermediate or a substrate in a biosynthesis system of a target substance. For example, a method of producing L-lysine by utilizing a Corynebacterium bacterium strain in which expression of the L-lysine export gene (lysE) is enhanced have been reported (WO97/23597). Furthermore, a method of producing an L-amino acid using a microorganism in which expression of the rhtA, B, and C genes are enhanced has also been reported (European Patent Publication No. 1013765). These genes have been reported to be involved in the export of L-amino acids. As a method of eliminating an export system of an intermediate or a substrate in a biosynthesis system of L-glutamic acid, mutating or disrupting a 2-oxoglutarate permease gene to reduce 2-oxoglutarate export, which is an intermediate of L-glutamic acid biosynthesis, is known (WO97/23597).
Furthermore, a method of breeding microorganisms has been suggested (WO00/37647) in which amino acid transport via the cell membrane is modified using a gene encoding the ATP binding cassette superfamily (ABC transporter), which is involved in the permeation of substances via a cell membrane.
In coryneform bacteria, it has been reported that addition of biotin or a surfactant changes the permeability of cell membranes, and thereby L-glutamic acid is exported from inside of the cells, which suggests that the export of L-glutamic acid in coryneform bacteria is not mediated by any export gene (Eiichiro Kimura, Metabolic Engineering of Glutamate Production, Advanced Biochemical Engineering Biotechnology, 79:37-57, 2003, Springer Verlag). Furthermore, it has also been reported that L-glutamic acid production efficiency was improved in Escherichia bacteria by enhancing the expression of a yfiK gene, which is thought to be involved in L-amino acid export (European Patent Publication 1016710).
However, an L-glutamic acid export gene has not been reported for a Pantoea microorganism or other microorganisms, and discovery of a novel L-glutamic acid export gene is desired.
Alternatively, a method of culturing a microorganism to produce and precipitate L-glutamic acid under acidic conditions is known (European Patent Publication 1078989). Enterobacter bacteria in which 2-oxoglutarate dehydrogenase activity is eliminated or reduced are often used in the fermentative production of precipitation of L-glutamic acid (European Patent Publication 1078989). In general, L-glutamic acid is converted into a TCA cycle intermediate, 2-oxoglutarate, in one step by glutamate dehydrogenase after it is imported into cells, and therefore it is generally considered that L-glutamic acid which is imported into cells is easily metabolized. However, when a microorganism having inactivated or reduced 2-oxoglutarate dehydrogenase activity is cultured under conditions for L-glutamic acid precipitation, the ratio of free L-glutamic acid having no electric charge becomes high, and readily passes through cell membranes, resulting in an increase in the intracellular L-glutamic acid concentration, and thus a decrease in bacterial cell growth. In European Patent Publication 1078989, a 2-oxoglutarate dehydrogenase-deficient strain which can efficiently produce and precipitate L-glutamic acid was bred by mutation treatment and used for the production of L-glutamic acid. However, few strains have been reported other than the above strain which can produce L-glutamic acid while also precipitating it, and no gene has been reported that can impart to a host microorganism L-glutamic acid resistance and L-glutamic acid-producing ability under conditions for precipitating L-glutamic acid.
The yhfK gene is a gene which exists on the genome of Escherichia coli (Science, 277(5331):1453-74, 1997), and it has been reported that it encodes a putative transporter based of the motifs, topology etc. of the predicted amino acid sequence (J. Mol. Microbiol. Biotechnol., 2 (2):195-198, 2000). However, neither cloning nor expression analysis of the gene has been previously reported. Furthermore, the actual function of the gene remains unknown.